We are proposing to investigate the effects of non-nucleoside inhibitor-resistance (NNRTI-R) mutations on HIV-1 replication and reverse transcriptase function. During the current funding period, we have demonstrated that 4 NNRTI-R mutants of HIV-1 (K103N, V106A, Y181C, and P236L) have characteristic effects on RNase H activity of HIV-1 RT. We have also demonstrated that NNRTI-R mutants with greater reductions in RNase H activity impair replication fitness in cell culture. Mutants that have greater abnormalities in RNase H activity also appear to have a reduced likelihood of appearing during clinical failure of NNRTIs. The RNase H cleavage abnormalities that we have described affect not only non-specific RNase H cleavages that degrade the viral RNA genome, but also the site-specific cleavages that form the polypurine tract (PPT), which serves as the primer for plus-strand synthesis. These findings are quite unexpected, since NNRTI-R mutations are in the polymerase domain, remote from the RNase H active site. Further study of these mutants should lead to a better understanding of (1) the pathogenic consequences of selection for NNRTI-R mutants, (2) the effects of RNase H cleavage on specific steps in reverse transcription and their contribution to viral replication fitness, and (3) how RNase H cleavages are modulated by residues in the polymerase domain of RT. We propose to pursue these important questions with the following specific aims: 1. Determine the effects of NNRTI-R mutants on specific steps in reverse transcription. 2. Determine the mechanisms by which NNRTI-R mutations affect RNase H cleavage. 3. Identify the mechanisms by which clinical isolates of HIV- 1 can compensate for the reduced replication fitness conferred by NNRTI-R mutations. 4. Determine the factors that contribute to selection for secondary NNRTI-R mutations during therapy with efavirenz. In order to accomplish these specific aims, we will utilize a broad array of experimental approaches, ranging from studies of RT structure and function in vitro, to analyses of the replication characteristics and biochemical function of HIV- 1 from patient samples.